Although spermatogonial stem cells are essential for reproduction, their regulation is poorly understood. Environmental, genetic and epigenetic factors are crucial for stem cell establishment, maintenance, and function, but systematically identifying these factors is extremely challenging in mammalian systems. Consequently, the behavior of most stem cells within their natural tissue microenvironments, or niches, is not well-understood. However, understanding stem cell-niche interactions is critical for developing successful stem cell-based therapeutic approaches. We use Drosophila spermatogenesis as a model system, since it parallels mammalian systems, yet we can precisely locate the stem cells and manipulate their niche genetically. Prior funding enabled us to discover the molecular mechanism controlling stem cell renewal in the Drosophila testis. Stem cells adhere to a small cluster of somatic support cells called the hub, which secretes a ligand that activates Janus-kinase-signal transducer and activator of transcription (Jak-STAT) signaling in adjacent cells, instructing them to remain as stem cells. Daughters displaced away from the hub differentiate. Building on these findings, in this renewal we will characterize additional factors we have recently found to be required for stem cell maintenance in this niche, and determine if they act together with the Jak-STAT signaling pathway during this process. This includes using a combination of genetic and biochemical approaches to assess the role of 1) chromatin remodeling complexes and 2) hormonal signaling pathways in spermatogonial stem cell renewal. We will also identify additional genes regulated by Jak-STAT signaling then use genetic approaches to determine their roles in both normal and aged niches. Together this work will provide fundamental insight into the genetic and epigenetic mechanisms that regulate stem cell maintenance. PUBLIC HEALTH RELEVANCE: This work will contribute significantly to what is known about the mechanisms regulating stem cells within an intact stem cell microenvironment (or niche) in vivo. Understanding how stem cells respond to niche signals is of fundamental importance for developing successful strategies to manipulate these potent cells in regenerative medicine.